Electroactive biopolymer optical and electro-optical devices and method of manufacturing the same

ABSTRACT

A method of manufacturing a biopolymer optical device includes providing a polymer, providing a substrate, casting the polymer on the substrate, and enzymatically polymerizing an organic compound to generate a conducting polymer between the provided polymer and the substrate. The polymer may be a biopolymer such as silk and may be modified using organic compounds such as tyrosines to provide a molecular-level interface between the provided bulk biopolymer of the biopolymer optical device and a substrate or other conducting layer via a tyrosine-enzyme polymerization. The enzymatically polymerizing may include catalyzing the organic compound with peroxidase enzyme reactions. The result is a carbon-carbon conjugated backbone that provides polymeric “wires” for use in polymer and biopolymer optical devices. An all organic biopolymer electroactive material is thereby provided that provides optical functions and features.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/940,168, filed on Jul. 11, 2013, which is a Continuation of U.S.patent application Ser. No. 12/513,392, filed on May 4, 2009, which isnow U.S. Pat. No. 8,574,461, which is a National Stage Entry ofPCT/US2007/083639, entitled “ELECTROACTIVE BIOPOLYMER OPTICAL ANDELECTRO-OPTICAL DEVICES AND METHOD OF MANUFACTURING THE SAME” filed Nov.5, 2007, which claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 60/856,297 filed on Nov. 3, 2006, entitled“Biopolymer Devices and Methods for Manufacturing the Same.”

GOVERNMENT SUPPORT

The invention was made with government support under grant numberFA95500410363 awarded by the Air Force Office of Scientific Research.The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is directed to electroactive biopolymer opticaland electro-optical devices and methods for manufacturing such devices.The present invention is further directed to electronics and electricalintegration to biopolymer optical and electro-optical devices.

Description of Related Art

The field of optics is well established. Some subfields of opticsinclude diffractive optics, micro-optics, photonics and guided waveoptics. Various optical devices have been fabricated in these and othersubfields of optics for research and commercial application. Forexample, common optical devices include diffraction gratings, photoniccrystals, optofluidic devices, waveguides, lenses, microlens arrays,pattern generators, beam reshapers, and the like.

These optical devices are fabricated using various methods depending onthe application and the optical characteristics desired. However, theseoptical devices, and the fabrication methods employed in theirmanufacture, generally involve significant use of non-biodegradablematerials. For example, glass, fused silica, and plastic are commonlyused in optical devices. Such materials are not biodegradable and remainin the environment for extended periods of time after the opticaldevices are removed from service and discarded. Of course, some of thematerials can be recycled and reused. However, recycling also requiresexpenditures of natural resources and adds to the environmental costsassociated with such materials.

Therefore, there exists an unfulfilled need for optical devices thatminimize the negative impact to the environment. In addition, thereexists an unfulfilled need for optical devices that provide additionalfunctional features that are not provided by conventional opticaldevices.

SUMMARY OF THE INVENTION

In view of the foregoing, objects of the present invention are toprovide various novel biopolymer optical devices and methods formanufacturing such optical devices that may be used in variousapplications.

One aspect of the present invention is to provide electroactivebiopolymer optical and electro-optical devices.

Another aspect of the present invention is to provide a method formanufacturing such biopolymer optical devices.

One advantage of the present invention is in providing biopolymeroptical devices that minimize the negative impact to the environment.

Another advantage of the present invention is in providing biopolymeroptical devices that are biocompatible.

Yet another advantage of the present invention is in providingbiopolymer optical devices that have additional functional features thatare not provided by conventional optical devices.

In the above regard, inventors of the present invention recognized thatbiopolymers, and especially silk proteins, present novel structure andresulting functions. For example, from a materials science perspective,silks spun by spiders and silkworms represent the strongest and toughestnatural fibers known and present various opportunities forfunctionalization, processing, and biocompatibility. Over five millenniaof history accompany the journey of silk from a sought-after textile toa scientifically attractive fiber. As much as its features captivatedpeople in the past, silk commands considerable attention in this day andage because of its strength, elasticity, and biochemical properties. Thenovel material features of silks have recently been extended due toinsights into self-assembly and the role of water in assembly. Theseinsights, in turn, have led to new processing methods to generatehydrogels, ultrathin films, thick films, conformal coatings,three-dimensional porous matrices, solid blocks, nanoscale diameterfibers, and large diameter fibers.

Silk-based materials achieve their impressive mechanical properties withnatural physical crosslinks of thermodynamically stable proteinsecondary structures also known as beta sheets (β-sheets). As such, noexogenous crosslinking reactions or post process crosslinking isrequired to stabilize the materials. The presence of diverse amino acidside chain chemistries on silk protein chains facilitates couplingchemistry for functionalizing silks, such as with cytokines, morphogens,and cell binding domains. There are no known synthetic orbiologically-derived polymer systems that offer this range of materialproperties or biological interfaces, when considering mechanicalprofiles, aqueous processing, room-temperature processing, ease offunctionalization, diverse modes of processing, self-forming crosslinks,biocompatibility, and biodegradability.

Another unique feature provided by the biopolymer devices in accordancewith the present invention, and especially the silk proteins, is theability to genetically alter the native sequence of the biopolymer toadd new functions, or to chemically modify the biopolymer to add newfunctions. The method and biopolymer devices of the present inventionextend the capabilities of added cell binding domains (RGD), redoxtriggers (methionines for oxidation/reduction control), andphosphorylation triggers (enzymatic kinase/phosphatase reactions). Thebiopolymer optical devices of the present invention further geneticallyredesign new versions of silk that retain native silk assembly andstructure, but offer additional functions.

Additionally, using methods of the present invention, a variety ofaromatic organic compounds, including tyrosines, can be enzymaticallypolymerized to generate conducting polymers. The polymerization of theorganic compounds may be performed from solutions or in the solid state.This enzymatic process may be catalyzed by peroxidase enzyme reactionsand is based on free radical coupling. The result is a carbon-carbonconjugated backbone that provides polymeric “wires” for use in polymerand biopolymer optical devices.

Further modifications to biopolymers such as silk may be made withtyrosines, either genetically or via chemical coupling. Tyrosinesprovide a molecular-level interface between the bulk silk protein andthe optical features with a conducting layer or features viatyrosine-enzyme polymerization. Correspondingly, a unique, all-organicbiopolymer electroactive material may be realized that also providesoptical features.

More specifically, in accordance with the present invention, tyrosinemonomers can be enzymatically crosslinked to form conducting polymers.The optical gratings made of biopolymers such as silk may bere-engineered to genetically encode tyrosine blocks in the silk.Tyrosine crosslinking may be used to form conducting wires, and furthercontrols may be implemented to control the position at which the “wires”are formed, both internally and on the surface of the silk. As such, thepresent invention provides directed integration of electronic componentsinto the biopolymer optical devices. These biopolymer materials,including silk, can be used for electronic properties for new conformalcoatings and related technologies and include additional opticalfeatures.

The tyrosine moieties may be incorporated in the polymer or biopolymersuch as silk via genetic engineering or via surface chemistry as a“functional” fusion component. For example, carbodiimide coupling may beused to incorporate the tyrosine moieties. Subsequent post processingpolymerization via enzymatic processes generates conjugated conduitsalong the silk protein assemblies. The polymerization step is based on asecondary enzymatic polymerization with peroxidase to stitch thetyrosine carbon to carbon (C to C) bonds together to generate conductingpolymers. The ability to form nanolayers, nanofibers, and relatedmaterial systems with precise control of conducting polymer location andfeatures provides new options for forming conformal, light weight,functional protective coatings with enhanced electronic and opticalfunctions for a variety of applications.

Peroxidase catalysis, mediated by hydrogen peroxide, was used to formconducting polymers from a wide range of aromatic compounds. Horseradishperoxidase (HRP) is a glycoprotein that contains a single-chain β-typehemoprotein with an Fe containing porphyrin. HRP catalysis of aromaticcompounds was used to form the conducting polymers. The solid-statepolymerization reactions of aromatics on surfaces, via peroxidasecatalyzed reactions, was used to form conducting polymers. In oneembodiment of the present invention, dip-pen nanolithography (DPN) wasused to pattern 4-aminothiophenol and tyrosines as the “ink”. DPNpatterning of an aromatic monomer, with surface induced orientation, wasused to promote enzymatic polymerization under ambient surface reactionsto form conducting polymers.

An example surface reaction may include a 0.01 M H₂O₂ stock solution,prepared by diluting H₂O₂ water solution (30% w/w) with MeOH/H₂O (1:1 byvolume) mixture. Peroxidase or hematin catalyzed polymerization may becarried out by immersing the solid state assemblies (eitherself-standing or on the surface of glass slides) into the H₂O₂ stocksolution, which contains 200 μL horseradish peroxidase stock solution.The silk assembly is washed by dipping it in buffer solutions severaltimes after the reaction. The peroxidase (donor: hydrogen peroxideoxidoreductase; EC 1.11.1.7, Type II, from horseradish, and hematin(procine) are commercially available.

Hematin provides benefits in the solid state material reactions due tothe smaller size of the molecule compared to horseradish peroxidase,which relates to diffusion of the tyrosines not at the surface of thebulk materials, for example, with internal blocks. A typical hematinreaction includes sodium phosphate buffer, the silk material, andhematin. An equal molar amount of hydrogen peroxide (0.6 mmol) is addedas oxidant, as in the peroxidase reactions.

Various applications exist for the electroactive biopolymer devices inaccordance with the present invention. For example, the electroactivebiopolymer devices may be used as electro-optical collectors, solarcollectors, mechanical actuators with optical readout, and in otherapplications where light-weight, degradable, electroactive devices aredesired.

While no other biopolymer or synthetic polymer can match the range offeatures outlined above for silk, the inventors of the present inventionhave identified some other polymers that exhibit various propertiessimilar or analogous to silk. In particular, other natural biopolymersincluding chitosan, collagen, gelatin, agarose, chitin,polyhydroxyalkanoates, pullan, starch (amylase amylopectin), cellulose,hyaluronic acid, and related polymers have been identified. In view ofthe above-noted features of biopolymers and of silk in particular, thepresent invention provides novel biopolymer optical devices and methodsfor manufacturing such devices.

In accordance with one aspect of the present invention, a method ofmanufacturing a conducting polymer includes providing a polymer,providing a substrate, casting the polymer on the substrate, andenzymatically polymerizing an organic compound to generate a conductingpolymer between the provided polymer and the substrate. In oneembodiment, the polymer provided is a biopolymer, such as silk, while inother embodiments, other polymers may be used including chitosan,collagen, gelatin, agarose, chitin, polyhydroxyalkanoates, pullan,starch (amylose amylopectin), cellulose, hyaluronic acid,polydimethylsiloxane (PDMS), and related biopolymers, or variations andcombinations thereof.

In one embodiment, the polymer may be a matrix solution, and thepolymerization is performed from the matrix solution, while in otherembodiments, the polymer may be a solid, and the polymerization isperformed from the solid. Additionally, in one embodiment, the enzymaticpolymerization may include catalyzing the organic compound withperoxidase enzyme reactions. For example, in one embodiment, tyrosine isused as the organic compound, while in other embodiments, other organiccompounds may be used including red blood cells, horseradish peroxidase,phenolsulfonphthalein, nucleic acid, a dye, a cell, an antibody,enzymes, for example, peroxidase, lipase, amylose, organophosphatedehydrogenase, ligases, restriction endonucleases, ribonucleases, DNApolymerases, glucose oxidase, laccase, cells, viruses, proteins,peptides, small molecules (e.g., drugs, dyes, amino acids, vitamins,antioxidants), DNA, RNA, RNAi, lipids, nucleotides, aptamers,carbohydrates, chromophores, light emitting organic compounds such asluciferin, carotenes and light emitting inorganic compounds (such aschemical dyes), antibiotics, antifungals, antivirals, light harvestingcompounds such as chlorophyll, bacteriorhodopsin, protorhodopsin, andporphyrins and related electronically active compounds, or variationsand combinations thereof.

In one embodiment of the present invention, an interface is formedbetween the polymer and the substrate, where the interface is aconducting layer formed by tyrosine-enzyme polymerization. The interfacemay include a carbon to carbon (C to C) conjugated backbone.

In one embodiment, the substrate may act as a mold or a template for anoptical device. The substrate may be a mold or template forelectro-optical collectors, solar collectors, mechanical actuators withoptical readout, and other applications where light-weight, degradable,electroactive devices are desired. The substrate may also be a mold oftemplate for biopolymer devices including lenses, microlens arrays,optical gratings, pattern generators, beam reshapers, and the like.Other geometric features, such as holes and pits, for example, may alsobe included in the substrate.

In accordance with another embodiment of the present invention, aconducting polymer is provided that is made of at least a bulk protein,a substrate, an organic compound, and an enzyme that polymerizes theorganic compound to generate a conducting polymer between the polymerand the substrate. The conducting polymer may be a biopolymer, such assilk, or may be another polymer including chitosan, collagen, gelatin,agarose, polyhydroxyalkanoates, pullan, starch (amylose amylopectin),cellulose, hyaluronic acid, polydimethylsiloxane (PDMS), and relatedbiopolymers, or combinations thereof.

These and other advantages and features of the present invention willbecome more apparent from the following detailed description of thepreferred embodiments of the present invention when viewed inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic flow diagrams illustrating a method ofmanufacturing a biopolymer optical device in accordance with oneembodiment of the present invention.

FIG. 2 is a graph that illustrates the relationship between the volumeof 8% silk concentration vs. film thickness.

FIG. 3A is a photograph of a biopolymer film made of silk.

FIG. 3B is a graph showing the prism coupled angular dependence ofreflectivity of the biopolymer film of FIG. 8A.

FIG. 3C is a graph showing the measured transmission of light throughthe biopolymer film of FIG. 8A.

FIG. 4 illustrates results graphs showing retention of the hemoglobinfunction within an RBC-doped silk optical device.

FIG. 5 is a photograph showing diffractive biopolymer devices that havebeen cast in silk, chitosan, and collagen.

FIG. 6 is a graph showing antibody IgG1 activity related to initialactivity in the silk films prepared in the two different formats andstored at the three different temperatures.

FIG. 7 is a graph showing antibody IgG activity related to initialactivity in the silk films prepared in the two different formats andstored at the three different temperatures.

DETAILED DESCRIPTION OF THE INVENTION

In view of the superior functional characteristics and processabilitynoted above, the biopolymer optical devices in accordance with thepresent invention are described as being fabricated using a biopolymersuch as silk. In this regard, the silk utilized was silkworm silk.However, there are many different silks, including spider silk,transgenic silks, and genetically engineered silks, variants andcombinations thereof and others, that may alternatively be used tomanufacture biopolymer optical devices in accordance with the presentinvention.

In addition, other biodegradable polymers may be used instead of silk.For example, other biopolymers, such as chitosan, exhibit desirablemechanical properties, can be processed in water, and form generallyclear films for optical applications. Other biopolymers, such ascollagen, cellulose, chitin, hyaluronic acid, amylase, and the like mayalternatively be utilized in specific applications. Syntheticbiodegradable polymers such as polyactic acid, polyglycolic acid,polyhydroxyalkanoates, and related copolymers may also be selectivelyused. Such polymers may be used by themselves, or in combination withsilk and other polymers and may be used to manufacture biopolymeroptical devices for specific applications.

FIG. 1A is a schematic illustration of a flow diagram 10 showing amethod of manufacturing a biopolymer optical device in accordance withone embodiment of the present invention. If a biopolymer is provided instep 11, the process proceeds to step 16 below. Otherwise, a biopolymeris provided in step 12. In the example where the biopolymer is silk, thebiopolymer may be provided by extracting sericin from the cocoons ofBombyx muni. In one embodiment, the biopolymer may be a solution such asa biopolymer matrix solution, while in other embodiments, differentsolvents other than water, or a combination of water and other solventsmay be used, depending on the biopolymer used.

In the example of silk, an aqueous silk fibroin solution may beprocessed, for example, 8.0 wt %, which is then used to manufacture thebiopolymer optical device. Of course, in other embodiments, the solutionconcentrations may also be varied from very dilute (approximately 1 wt%) to very high (up to 30 wt %) using either dilution or concentration,for example, via osmotic stress or drying techniques. In this regard,other embodiments may utilize different percent weight solutions tooptimize flexibility or strength of the resultant nanopatternedbiopolymer optical device, depending on the application. Production ofaqueous silk fibroin solution is described in detail in WIPO PublicationNumber WO 2005/012606 entitled “Concentrated Aqueous Silk FibroinSolution and Uses Thereof,” which is incorporated by reference.Additionally, the polymer may be a solid, and the polymerization is thenperformed using the solid.

A substrate is provided in step 16 to serve as a mold or template inmanufacturing the biopolymer optical device. A surface of the substratehas the desired characteristic features to be formed on the biopolymeroptical device. In this regard, the substrate may be an appropriatenanopattern on a surface of the optical device and may be an opticaldevice such as a nanopatterned optical grating or other optical device,depending on the optical features desired for the device beingmanufactured. The polymer, such as the aqueous biopolymer matrixsolution or the solid described above, is cast on the substrate in step18. Upon drying, and upon completion of the subsequent reactions, asolidified biopolymer film is formed on the surface of the substrate.The thickness of the biopolymer film depends on the volume of thebiopolymer matrix solution or the solid polymer applied to thesubstrate.

Patterned nanostructures can be provided on the biopolymer films, suchas the silk films manufactured in the above discussed manner. In oneembodiment, the surface of the substrate may be smooth so as to providea smooth biopolymer film, and a nanopattern may be machined on thesurface of the biopolymer film. The nanopattern may be machined using alaser, such as a femtosecond laser, or by other nanopattern machiningtechniques, including lithography techniques such as photolithography,electron beam lithography, and the like. Using such techniques,nanopattern features as small as 700 nm that are spaced less than 3 μmhave been demonstrated as described in further detail below.

In another embodiment, the surface of the substrate itself may have anappropriate nanopattern thereon so that when the solidified biopolymerfilm is removed from the substrate, the biopolymer film is alreadyformed with the desired nanopattern on a surface thereof. In such animplementation, the substrate may be an optical device such as ananopatterned optical grating, depending on the nanopattern desired onthe biopolymer films. The substrate surfaces may be coated with Teflon™and other suitable coatings to ensure even detachment after thebiopolymer matrix solution transitions from the liquid to the solidphase. The ability of the biopolymer casting method using ananopatterned substrate for forming highly defined nanopatternedstructures in the resultant biopolymer films was verified, and silkfilms having nanostructures as small as 75 nm and RMS surface roughnessof less than 5 nm have been demonstrated.

Referring again to FIG. 1A, in step 20, an organic compound isenzymatically polymerized to generate a conducting polymer between theprovided polymer and the substrate. The enzymatic reaction geneticallyalters the native sequence of the silk protein to add new functions orchemically modifies the biopolymer to add new functions, depending uponthe polymer (for example, the silk protein) chosen and the enzymereaction components. The method of the present invention extends thecapabilities of added cell binding domains (Arginine-Glycine-Asparticacid—RGD), redox triggers (methionines for oxidation/reduction control),and phosphorylation triggers (enzymatic kinase/phosphatase reactions).The enzymatic polymerization of the silk proteins of the presentinvention further genetically redesigns new versions of silk that retainnative silk assembly and structure, but offer additional functions.

In one embodiment, the polymerized organic compound may be aromaticorganic compounds such as amino acids, including tyrosines, that can beenzymatically polymerized to generate conducting polymers. As indicatedabove, the polymerization of the organic compounds may be performed froma solution or from a solid state.

Further modifications to biopolymers such as silk may be made withtyrosines, either genetically or via chemical coupling. Tyrosinesprovide a molecular-level interface between the bulk silk protein andthe optical features with a conducting layer or with features viatyrosine-enzyme polymerization. Correspondingly, a unique, all-organicbiopolymer electroactive material may be realized that also providesoptical features.

More specifically, as shown in step 210, tyrosine monomers can beenzymatically crosslinked to form conducting polymers. The opticalgratings made of biopolymers such as silk may be re-engineered togenetically encode tyrosine blocks in the silk as shown in step 212.

As shown in step 22 in FIG. 1B, the enzymatic process may be catalyzedby peroxidase enzyme reactions and is based on free radical coupling.For example, peroxidase catalysis, mediated by hydrogen peroxide, wasused to form conducting polymers from a wide range of aromaticcompounds. Horseradish peroxidase (HRP) is a glycoprotein that containsa single-chain β-type hemoprotein with an Fe containing porphyrin. HRPcatalysis of aromatic compounds was used to form the conductingpolymers. The solid-state polymerization reactions of aromatics onsurfaces, via peroxidase catalyzed reactions, was used to formconducting polymers. In one embodiment of the present invention, dip-pennanolithography (DPN) was used to pattern 4-aminothiophenol andtyrosines as the “ink”. DPN patterning of an aromatic monomer, withsurface induced orientation, was used to promote enzymaticpolymerization under ambient surface reactions to form conductingpolymers.

The tyrosine moieties can be incorporated in the biopolymer such as silkvia genetic engineering or via surface chemistry as a “functional”fusion component. For example, carbodiimide coupling may be used toincorporate the tyrosine moieties. Subsequent post processingpolymerization via enzymatic processes generates conjugated conduitsalong the silk protein assemblies. As shown in step 214, thepolymerization step is based on a secondary enzymatic polymerizationwith peroxidase to stitch the tyrosine carbon to carbon (C to C) bondstogether to generate conducting polymers. The ability to formnanolayers, nanofibers, and related material systems with precisecontrol of conducting polymer location and features provides new optionsfor forming conformal, light weight, functional protective coatings withenhanced electronic and optical functions for a variety of applications.

An example surface reaction may include a 0.01 M H₂O₂ stock solution,prepared by diluting H₂O₂ water solution (30% w/w) with MeOH/H₂O (1:1 byvolume) mixture. Peroxidase or hematin catalyzed polymerization can becarried out by immersing the solid state assemblies (eitherself-standing or on the surface of glass slides) into the H₂O₂ stocksolution which contains 200 μL horseradish peroxidase stock solution.The silk assembly is washed by dipping it in buffer solutions severaltimes after the reaction. The peroxidase (donor: hydrogen peroxideoxidoreductase; EC 1.11.1.7), Type II, from horseradish, and hematin(procine) are commercially available.

Hematin provides benefits in the solid state material reactions due tothe smaller size of the molecule compared to horseradish peroxidase,which may relate to diffusion of the tyrosines not at the surface of thebulk materials, for example, with internal blocks. A typical hematinreaction includes sodium phosphate buffer, the silk material, andhematin. An equal molar amount of hydrogen peroxide (0.6 mmol) would beadded as oxidant, as in the peroxidase reactions.

As shown in step 24, tyrosine crosslinking may be used to formconducting polymeric “wires” for the biopolymer optical device,resulting from a carbon-carbon (C to C) conjugated backbone. As shown instep 36, further controls may be implemented to control the position atwhich the “wires” are formed, both internally and on the surface of thesilk. As such, directed integration of electronic components into thebiopolymer optical devices may be performed in accordance with thepresent invention. These biopolymer materials, such as silk, can be usedfor electronic properties for new conformal coatings and relatedtechnologies and include additional optical features.

Various applications of the electroactive biopolymer devices include useas electro-optical collectors, solar gelatin, mechanical actuators withoptical readout, and other applications where light-weight, degradable,electroactive devices can be used.

Experiments were conducted to validate the above-described method bymanufacturing various biopolymer optical waveguides. The relationshipbetween the volume of 8 wt % silk concentration aqueous silk fibroinsolution, and the resulting silk film thickness, is shown in the graph30 of FIG. 2, where the aqueous silk fibroin solution was cast over asubstrate surface of approximately 10 square centimeters. The X-axisshows the volume of silk fibroin solution in mL, and the Y-axis showsthe thickness of the resultant film in μm.

Of course, the film properties such as thickness and biopolymer content,as well as optical features, may be altered based on the concentrationof fibroin used in the process, the volume of the aqueous silk fibroinsolution or solid deposited, and the post-deposition process for dryingthe cast solution to lock in the structure. Accurate control of theseparameters is desirable to ensure the optical quality of the resultantbiopolymer optical waveguide and to maintain various characteristics ofthe biopolymer optical waveguide, such as transparency, structuralrigidity, and flexibility. Furthermore, additives to the biopolymermatrix solution may be used to alter features of the biopolymer opticalwaveguide such as morphology, stability, and the like, as known withpolyethylene glycols, collagens, and the like.

An unpatterned biopolymer film having a thickness of 10 μm wasmanufactured in the above-described manner using an aqueous silk fibroinsolution, and was characterized in a scanning prism coupledreflectometer from Metricon Corporation. FIG. 3A illustrates theunpatterned biopolymer film 34 manufactured and characterized. The indexof refraction of the biopolymer film 34 was measured to be n=1.55 at 633nm, which is slightly higher than the index of refraction ofconventional borosilicate glass. The measured index of refractionconfirms that the value is high enough to afford reasonable contrast foroptical use such as in air-silk biophotonic crystals (BPC)(Δn_(fibroin)−Δn_(air)=0.55). The characterization of the unpatternedsilk film 34 is shown in graph 36 of FIG. 3B, which clearly demonstratesthe prism coupled angular dependence of the reflectivity. Theoscillations in graph 36 are due to coupling into guided waves,demonstrating the use of silk as a waveguide material.

The measured roughness of cast silk film on an optically flat surfaceshows measured root mean squared roughness values between 2.5 and 5nanometers, which implies a surface roughness easily less than λ/50 at awavelength of 633 nm. Atomic force microscope images of patterned silkdiffractive optics show the levels of microfabrication obtainable bycasting and lifting silk films off of appropriate molds. The images showdefinition in the hundreds of nanometer range and the sharpness of thecorners indicates the possibility of faithful patterning down to thetens of nanometers.

In addition, the unpatterned silk film 34 was also analyzed to determinetransparency. FIG. 3C is a graph 38 that illustrates the measuredtransmission of light through the silk film 34 in various wavelengths.Transmission measurements indicate that the unpatterned silk film 34 washighly transparent across the visible spectrum. For comparison, similarthickness films were also cast in collagen, and polydimethylsiloxane(PDMS). The free-standing structural stability was found to be inferior,and the resultant biopolymer optical device was not self-supporting whenimplemented as a thin film. However, such biopolymers may be used inother applications if structural stability is deemed to be not asimportant.

Importantly, shaped films having various thicknesses were patterned onthe nanoscale using the method of FIG. 1 described above to providenanopatterned biopolymer optical devices.

The term “nanopatterned” as used with regard to the present inventionrefers to very small patterning that is provided on a surface of thebiopolymer optical device. The patterning has structural features whosesize can be appropriately measured on a nanometer scale (that is, 10⁻⁹meters), for example, sizes ranging from 100 nm to few microns.Additionally, the biopolymer optical devices of the present inventionmay incorporate various different optical devices such as lenses,diffraction gratings, photonic crystals, waveguides, and the like.

A variety of nanopatterned biopolymer optical devices were successfullymanufactured using the above-described method of the present inventionusing silk fibroin solution. These devices included waveguides, lenses,microlens arrays, optical gratings, pattern generators, and beamreshapers. In particular, the aqueous solution of silk fibroin was castonto specific substrates with patterns thereon. The substrate surfaceswere coated with Teflon™ to ensure even detachment after the biopolymermatrix solution transitions from the liquid to the solid phase. Theability of the biopolymer casting method of the present invention forforming highly defined nanopatterned structures in biopolymer opticaldevices was verified by casting the optical waveguides of the presentinvention. Regular patterned features with dimensions down to 210 nm,and localized surface roughness of less than 20 nm, have been attained.As mentioned above, smoothing techniques may also be used to furtherreduce or remove surface roughness of the biopolymer optical waveguide.

Such regular patterning of biocompatible materials allows manufacturingof optical devices that can be used to provide photonic bandgaps andmanipulate light via an organic, yet mechanically robust optical device.These devices combine the flexibility of embedded optics with the uniqueversatility of the protein substrate as explained throughout theapplication. Many advantages are provided by the present inventionincluding combining the organic nature of biopolymers such as silk withthe power of diffractive and transmissive optics embedded in an organicmatrix to create biologically active optical elements. Silk provides acontrollably degradable, biocompatible, and structurally strong mediumwith which to fabricate the optical devices in accordance with thepresent invention.

Transmissive nanopatterned diffractive biopolymer optical devices weremade using the method of the present invention described above. Theseoptical devices include biopolymer optical waveguides, silk diffusers,line pattern generators, and cross pattern generators. Such opticaldevices use appropriately configured wavelength scale surfacestructuring to create predefined one or two-dimensional light patternsthat exploit light interference. Such optical devices made ofconventional materials have been applied to imaging, spectroscopy, beamsampling and transformation, and metrology to name a few uses. Extendingthis approach to control the delivery of light within a biologicalmatrix such as silk biopolymer can provide optimal coupling of photonsinto a substrate or allow for designed optical discrimination,interface, or readout.

A significant advantage of biopolymer optical waveguides in accordancewith the present invention is the ability of the optical waveguides tobe biologically activated since they are entirely organic andbiocompatible. Water-based processing can be used, for example, for silkoptical waveguides. This increases cellular survivability of thewaveguides and the likelihood of biocompatibility.

To confirm biocompatibility of nanopatterned biopolymer optical devices,red blood cells (RBCs) were incorporated into a silk diffraction gratingin accordance with the present invention that was manufactured asdescribed above with regard to FIG. 1. The RBC-silk fibroin solution wasprepared by combining 1 ml of an 80% hematocrit human RBC solution and 5ml of the 8% silk solution. The mixture was cast on a 600 lines/mmoptical grating and allowed to dry overnight. The film was removed fromthe optical grating and annealed for two hours. The grating structurewas observed in the resultant RBC-doped silk diffraction grating.

The RBC-doped silk diffraction grating was then tested to observe thediffraction orders. An optical transmission experiment was performed todetermine whether hemoglobin (the oxygen-carrying protein contained inRBCs) maintained its activity within the matrix of the silk diffractiongrating. The results graphs 160 are shown in FIG. 4 and indicate theretention of hemoglobin function within the RBC-doped silk diffractiongrating. The X-axis corresponds to the wavelength (in nm), and theY-axis indicates the absorbance by the RBC-doped silk diffractiongrating.

In particular, the RBC-doped silk diffraction grating was inserted in aquartz cuvette filled with distilled water, and an absorbance curve wasobserved. This result is shown by line (b) HbO₂ in results graphs 160.As can be seen, the absorbance curve shown by line (b) HbO₂ exhibitedtwo peaks typical of oxy-hemoglobin absorption. Subsequently, nitrogengas was bubbled into the cuvette to deoxygenate the hemoglobin. After 15minutes, the characteristic absorption peaks of oxy-hemoglobindisappeared from the absorbance curve. This result is shown by line (a)Hb in the results graphs 160. These results were further confirmed whenthe nitrogen flow to the cuvette is subsequently halted, which resultedin the reappearance of the oxy-hemoglobin peaks. This result is shown byline (c) HbO₂ in results graphs 160.

As previously noted, alternative biopolymers may also be used forfabrication of nanopatterned biopolymer optical devices in accordancewith the present invention. FIG. 5 shows a photograph 180 thatillustrates other diffractive biopolymer optical devices that have beencast using different materials. In particular, a chitosan optical device182 and a collagen optical device 184 have also been manufactured inaccordance with the present invention. With respect to chitosan, opticaldiffraction characteristics similar to silk have been observed.

It should be evident from the above discussion and the examplenanopatterned biopolymer optical devices shown and discussed that thepresent invention provides biodegradable biopolymer optical devices.High quality biopolymer optical devices were manufactured that arenaturally biocompatible, can be processed in water, and can undergodegradation with controlled lifetimes. As explained above, thebiopolymer optical devices of the present invention may also bebiologically activated by incorporating small organic materials. Inparticular, the biopolymer optical devices can be biologicallyfunctionalized by optionally embedding it with one or more organicindicators, living cells, organisms, markers, proteins, and the like.More specifically, the biopolymer optical devices in accordance with thepresent invention may be embedded or coated with organic materials suchas red blood cells, horseradish peroxidase, phenolsulfonphthalein,nucleic acid, a dye, a cell, an antibody, as described further inAppendix I, enzymes, for example, peroxidase, lipase, amylose,organophosphate dehydrogenase, ligases, restriction endonucleases,ribonucleases, DNA polymerases, glucose oxidase, laccase, cells,viruses, proteins, peptides, small molecules (e.g., drugs, dyes, aminoacids, vitamins, antioxidants), DNA, RNA, RNAi, lipids, nucleotides,aptamers, carbohydrates, chromophores, light emitting organic compoundssuch as luciferin, carotenes and light emitting inorganic compounds(such as chemical dyes), antibiotics, antifungals, antivirals, lightharvesting compounds such as chlorophyll, bacteriorhodopsin,protorhodopsin, and porphyrins and related electronically activecompounds, tissues or other living materials, other compounds orcombinations thereof. The embedded organic materials are biologicallyactive, thereby adding biological functionality to the resultantbiopolymer optical device.

The embedding of the biopolymer optical devices with organic materialsmay be performed for example, by adding such materials to the biopolymermatrix solution used to manufacture the biopolymer films, such as thesilk fibroin matrix solution. In the implementation where the biopolymeroptical device is manufactured using a solid, the optical device can bebiologically functionalized by functionalizing of one or more of thepolymer films.

The present invention broadens the versatility of optical devices byallowing the direct incorporation of labile biological receptors in theform of peptides, enzymes, cells, antibodies, or related systems, andthe like and allows such optical devices to function as biologicalsensing devices.

The biopolymer optical devices of the present invention can be readilyused in environmental and life sciences where biocompatibility andbiodegradability are paramount. For example, the nanopatternedbiopolymer optical devices as described above can be unobtrusively usedto monitor a natural environment such as in the human body and may beimplanted in vivo without a need to retrieve the device at a later time.The degradation lifetime of the biopolymer optical devices of thepresent invention can be controlled during the manufacturing process,for example, by controlling the ratio and amount of the solution matrixcast or the type of polymer used. Moreover, the biopolymer opticaldevices of the present invention can be dispersed in the environment,again without the need to retrieve them at a later time, therebyproviding novel and useful devices for sensing and detection.

The foregoing description of the aspects and embodiments of the presentinvention provides illustration and description, but is not intended tobe exhaustive or to limit the invention to the precise form disclosed.Those of skill in the art will recognize certain modifications,permutations, additions, and combinations of those embodiments arepossible in light of the above teachings or may be acquired frompractice of the invention. Therefore the present invention also coversvarious modifications and equivalent arrangements that fall within thepurview of the appended claims.

Antibody Stability in Silk Films

Materials—Anti-IL-8 monoclonal antibody (IgG1) was purchased fromeBioscience, Inc. human polyclonal antibody IgG and human IgG ELISAQuantitation Kit were purchased from Bethyl Laboratories Inc. All otherchemicals used in the study were purchased from Sigma-Aldrich (St.Louis, Mo.).

Antibody entrapment in silk films—human polyclonal antibody IgG—Ten ml 1mg/ml IgG mixed with 167 ml 6% silk solution make the IgG concentrationin silk film mg/g silk. 100 μl of mixed IgG solution was added to eachwell of 96 well plate which was placed in a fume hood with cover openedovernight. The dried film was either treated or not treated withmethanol. For methanol treatment, the wells were immersed in 90%methanol solution for 5 min and dried in the fume hood. All dry 96 wellplates were then stored at 4° C., room temperature, and 37° C. (FIG. 6).

Anti-IL-8 monoclonal antibody (IgG1)—0.5 ml 1 mg/ml IgG1 mixed with 83ml 6% silk solution make the IgG1 concentration in silk film 0.1 mg/gsilk. 50 μl of mixed IgG1 solution was added to a well of 96 well platewhich was placed in a fume hood with cover opened overnight. The driedfilm was either treated or not treated with methanol. For methanoltreatment, the wells were immersed in 90% methanol solution for 5 minand dried in the fume hood. All dry 96 well plates were then stored at4° C., room temperature, and 37° C. (FIG. 7).

Antibody measurement—Five wells prepared at the same condition weremeasured for statistic. Pure silk (without antibody) was used as acontrol.

For non methanol-treated samples, 100 μl of PBS buffer, pH 7.4, wasadded to the well which was further incubated at room temperature for 30min to allow the film to completely dissolve. Aliquot of solution wasthen subjected to antibody measurement. For methanol-treated samples,100 μl HFIP was added into each well which was further incubated at roomtemperature for 2 hours to allow the film completely dissolve. The silkHFIP solution was dried in a fume hood overnight. The follow step wasthe same as non methanol-treated samples, added PBS buffer and pipettethe solution for antibody measurement.

ELISA—Polystyrene (96-well) microtitre plate was coated with 100 μL ofantigen anti-Human IgG-affinity at a concentration of 10 μg/mL preparedin antigen coating buffer (bicarbonate buffer, 50 mM, pH 9.6) and thenincubated overnight storage at room temperature. The wells were thenwashed three times with TBS-T buffer. The unoccupied sites were blockedwith 1% BSA in TBS (200 μL each well) followed by incubation for 30minutes at room temperature. The wells were then washed three times withTBS-T. The test and control wells were then diluted with 100 μL ofserially diluted serum. Each dilution was in TBS buffer. Seriallydiluted blanks corresponding to each dilution were also present. Theplate was then incubated for 1 h at room temperature. The plate waswashed again with TBS-T buffer (five times). Bound antibodies wereassayed with an appropriate conjugate of anti-human IgG-HRP (1:100,000),100 μL of it was coated in each well and kept at room temperature for 1hour. Washing of the plate with TBS-T (five times) was followed byaddition of 100 μL TMB in each well and incubation at room temperaturefor 5-20 min. The absorbance of each well was monitored at 450 nm on aVersaMax microplate reader (Molecular devices, Sunnyvale, Calif.).

1-26. (canceled)
 27. An electroactive biopolymer optical devicecomprising: a biopolymer having an optical feature; wherein thebiopolymer is a genetically altered biopolymer or a chemically modifiedbiopolymer comprising an aromatic organic compound; wherein thebiopolymer is characterized by an ability to form crosslinks between aplurality of aromatic organic compounds, and wherein the biopolymer ischaracterized as conductive at the aromatic organic compound crosslinks,so that the device has directed integration of electronic components.28. The device of claim 27, wherein the biopolymer is a geneticallyaltered or chemically modified silk polypeptide.
 29. The device of claim27, wherein the aromatic organic compound is tyrosine and the aromaticorganic compound crosslinks are crosslinks between at least two tyrosinemonomers.
 30. The device of claim 29, wherein the tyrosine crosslinksform a carbon-carbon conjugated backbone.
 31. The device of claim 29,wherein the tyrosine crosslinks form conjugated conduits along thebiopolymer.
 32. The device of claim 29, wherein the genetically alteredbiopolymer is a polypeptide whose sequence comprises at least onetyrosine block.
 33. The device of claim 27, wherein the crosslinks forminternal conductive wires or conductive wires on a surface of thebiopolymer.
 34. The device of claim 27, wherein a surface of thebiopolymer has a structure formed thereon, and wherein the structurecomprises one or more optical features.
 35. The device of claim 34,wherein the structure is a patterned structure.
 36. The device of claim35, wherein the patterned structure is a nanopatterned structure. 37.The device of claim 27, wherein the device is selected from the groupconsisting of an electro-optical collector, a solar collector, amechanical actuator with optical readout, a conformal coating, or afunctionally protective coating.
 38. The device of claim 27, wherein theone or more optical features is selected from a group consisting ofdiffraction gratings, photonic crystals, optofluidic devices,waveguides, lenses, microlens arrays, pattern generators, or beamreshapers.
 39. The device of claim 27, wherein the biopolymer is ananolayer, a nanofiber, or a film.
 40. The device of claim 27, whereinthe device is a textile.
 41. The device of claim 27, wherein thebiopolymer is a silk protein and the device is a coating for textiles.42. The device of claims 27, further comprising one or more smallorganic molecules.
 43. The device of claims 27, further comprisingorganic indicators, cells, organisms, markers, or proteins.
 44. Thedevice of claims 27, further comprising red blood cells, horseradishperoxidase, phenolsulfonphthalein, nucleic acids, a dye, a cell, anantibody, enzymes, restriction endonucleases, ribonucleases, DNApolymerases, glucose oxidase, laccase, tissues, cells, antibodies,viruses, proteins, peptides, small molecules, drugs, dyes, amino acids,vitamins, antioxidants, DNA, RNA, RNAi, lipids, nucleotides, aptamers,carbohydrates, chromophores, light emitting organic compounds,luciferin, carotenes, light emitting inorganic compounds, antibiotics,antifungals, antivirals, light harvesting compounds, chlorophyll,bacteriorhodopsin, protorhodopsin, porphyrins, or electronically activecompounds.